Recent advances in molecular biology in the study of the genomes of fossil organisms have challenged many ideas about the origins of humans. The study of ancient DNA, known as paleogenetics/paleogenomics, which examines the entire genome has revolutionized the means archaeologists have at their disposal to study the past and shake our image of sapiens along with their relatives. Moreover, thanks to technological advances, we are now able to see the appearance of our ancestors.
What is DNA and What are the Methods of Analysis?
The work of a geneticist consists in studying and analyzing the heritable characteristics of living beings (microorganisms, animals, plants, humans) by means of their genome, the entire genetic material of a living being, the carrier of which is DNA (deoxyribonucleic acid). This genetic material is passed on to each generation to ensure the production of organisms whose physical organization and physiology are largely the same as their parents. So when they analyze the DNA sequence of an individual, they have access to two important pieces of information: the genealogy and the morphological and physiological characteristics of the DNA.
DNA sequencing - the order in which nucleotide pairs follow each other - provides access to a wealth of information about the ancestors of the individuals studied and their origins as well as the demographic history of ancient populations.
DNA can be stored in the earth for thousands of years - less in hot climates. Its degradation over time reduces the endogenous (original) DNA present in the samples to a tiny amount. Moreover, because they are stored in the soil, they are often contaminated with environmental DNA; DNA from bacteria, fungi, viruses and other neighbouring organisms.
What Do the Paleogenetic Studies Show?
Since the first sequencing of a human genome in 2001 (Venter et al., 2001) (1), genetic research has progressed rapidly. With the complete sequencing of mitochondrial DNA (from the maternal lineage) - or mtDNA - taken from Neanderthals in 2008, palaeontology entered the era of genetics (Green et al., 2008). (2) In 2010, based on the mtDNA of an unidentified finger limb, paleogeneticists were able to determine that it had belonged to a female of a previously unknown population, the Denisovans. (3)
A few months later, the nuclear DNA from the paternal line of the Neanderthals is decoded, then in 2012 that of Denisova's woman, showing that the latter has a common ancestor with Neanderthals and sapiens (Neves, 2012). (4)
Genetics, then, informs us not only about human ancestry, but also about migration and interbreeding. Interbreeding, genetic variations, and also technologies and customs are revealed by certain genetic analyzes. In order to distinguish the evolution of humans and thus the different prehistoric periods, the periods are dated according to the emergence of new technologies and customs. Genetics shows that these technological developments, and thus the spread of ideas and many customs, are largely linked to the movements of populations around the world, with migrations and interbreeding.
DNA analyzes of samples from the Yamnaya population, a nomadic tribe from the Eurasian steppes that inhabited what is now Ukraine and Russia, have thus helped to explain the apparent cultural transfers between Eastern Europe and Western Europe, to trace the migration of the Yamnayas, and to shed light on the evolution of the genetic heritage of Europeans. When the Yamnayas arrived 5,000 years ago, the genome of Western Europeans resembled that of hunter-gatherers, descendants of the first Homo sapiens who arrived in Europe more than 45,000 years ago, but also of Middle Eastern farmers who arrived 7,500 years ago subsequently living in the area for 2,000 years. The arrival of the Yamama seems to have profoundly altered the genome of Northern Europeans, as it was almost 90% remodeled in England, for example. In addition to this, these groups of steppe herders carried a genetic variant that enabled them to digest lactose. Their migration to Europe during the Bronze Age, therefore, played a major role in the lactose tolerance of Europeans today.
While the European population was unable to do so after weaning, this genetic variant would have spread among them by the Middle Ages and would explain why up to 95% of the population of Northern Europe is lactose tolerant today, compared to only 25% of Italians.
Preservation of DNA
The DNA of our ancestors is rare. This organic molecule is fragile and can spoil easily, sometimes within a few hours. It is therefore the climatic properties that condition the preservation of the double helix. When temperature - or humidity - is high, usually no DNA remains. The presence of certain bones makes it easier to study DNA, such as the petrous bone, which contains the cochlea of the inner ear and isolates the DNA from the external environment. In order to alter the bones as little as possible, researchers must also reduce the amount of DNA needed for the studies.
Biotech startups are also facilitating paleogenetic studies through the use of DNA microchips. On these microchips, paleogeneticists synthesize and deposit small DNA fragments that are representative of genetic markers of modern humans and carry a fluorescent molecule. A single chip can contain more than a million different fragments. When they come into contact with the DNA fragments of a sample, the fragments that find their counterpart on the chip adhere to it. Now all that remains is to detect the characteristic sites of the markers present in the sample by fluorescence. Eva-Maria Geigl, a paleogeneticist at the Institut Jacques-Monod of the CNRS in Paris, explains: "For example, we can look for mutations in regions of the genome known for their variability, or for markers associated with phenotypes such as eye colour, even if we do not know much about them yet. The limitation of these microchips is that you can only find what you are looking for. ... " (Geigl, 2018). (5) When there is little or no DNA, they can sometimes perform proteomic analysis, which is the analysis of proteins contained in the remains. The proteins also carry genetic information. Less fragile than DNA, proteins accumulate in the bones and enamel of the teeth, where they are protected by the mineral matrix. They can survive in this particular spot for more than two million years; they also allow identification by a protein present in the teeth, encoded by a gene from the X and Y chromosomes, which tells us the sex of the person among other information. However, if we can find a hundred proteins up to a few tens of thousands of years, only two proteins of the collagen family remain after that, which limits research to certain periods.
A Teamwork: Paleogenetics and Technology
Recently, on September 14, 2021, Parabon NanoLabs, an American company specializing in DNA technology and a leader in forensic DNA chip analysis, revealed the predicted faces of three ancient mummies from Abusir el-Meleq in Egypt at the 32nd International Symposium on Human Identification for the first time in Orlando, Florida. The mummy samples, estimated to be between 2,023 and 2,797 years old, were processed by researchers at the Max Planck Institute for the Study of Human History and the College of Tübingen in Germany (Schuenemann et al. 2017). (6)
To examine the ancient DNA contained in the mummy samples, enzymatic damage repair was performed on each sample. The DNA samples were then sequenced using a capture assay targeting 1.24 million single nucleotide polymorphisms (SNPs) and aligned to the human reference genome.
This allowed Parabon to generate whole-genome sequencing data and make it available to the public in the European Nucleotide Archives (ENA). From this whole-genome database, three samples with the highest data quality were selected for analysis. According to Parabon Labs, this appears to be the first time that whole human DNA phenotyping has been performed using such old samples.
This type of work was made possible by recent advances in bioinformatics in the field of low-coverage imputation, which allows for the highly accurate determination of common SNP genotypes from low-coverage sequencing data. This new imputation technology could now enable biotech companies like Parabon NanoLabs to work on even the most difficult, ancient or forensic samples.
The Snapshot DNA phenotyping pipeline was applied to each of the three mummy samples. It was specifically calibrated to handle missing data and predicted the ancestry, pigmentation, and facial morphology of each mummy. Interestingly, their respective ancestry proves to bear more resemblance to the modern Mediterranean and Middle Eastern people than to modern Egyptians. Their complexion must have been light brown, with dark eyes and hair and no freckles. These results are largely consistent with the findings of Schuenemann, who noted: "Ancient Egyptians had more ancestors from the Middle East than modern Egyptians, who received additional admixture from the sub-Saharan region only more recently, and that they had an allele for lighter skin in their DNA." (Schuenemann et al 2017) (6)
Three-dimensional facial morphology was derived by predicting the values of the major facial components, which were then converted into 3D graphical meshes. Facial predictions were compared, and heat maps were calculated to show differences between the subjects. These differences were then highlighted to create cartoon-like faces, which were combined with pigmentation predictions to create compositions of the likely appearance of the subjects aged 25 by forensic artist Parabon.
Announcing the results of the study, Dr Ellen Greytak, Director of Bioinformatics at Parabon, said:
"It's great to see how genome sequencing and advanced bioinformatics can be applied to ancient DNA samples (...) Just as in the case of Parabon's charge, these techniques revolutionize ancient DNA analysis by working with fragmented DNA." (7)
Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., et al. The sequence of the human genome. Science n° 291, 2001.
Green, R., Malaspinas A-S., et al. A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing quote. Cell, 2008 https://www.cell.com/fulltext/S0092-8674(08)00773-3
Krause, J., Fu, Q., Good, J.M., Viola, B., Shunkov, M. Anatoli P. Derevianko et Svante Pääbo, « The complete mitochondrial DNA genome of an unknown hominin from southern Siberia » [archive], Nature, 2010.
Neves, A., Serva, M. Extremely Rare Interbreeding Events Can Explain Neanderthal DNA in Living Humans, 2012. https://doi.org/10.1371/journal.pone.0047076
Eva-María Geigl. "Contribution de la paléogénétique à l’archéologie. Bioarchéologie: minimums méthodologiques", référentiels communs et nouvelles approches, 2019.
Schuenemann, V. J. et al. Ancient Egyptian mummy genomes suggest an increase of Sub-Saharan African ancestry in post-Roman periods. Nat. Commun n°8, 2017.